The objective of this revised VA Merit Proposal is to design CD8+ T cell-targeted pathogen- free (subunit) vaccines against infectious diseases and to devise an efficacious method for their delivery to mucosal sites, which mark the major port of pathogen entry. Most currently used vaccines work by antibody-mediated neutralization and/or opsonization. Despite the proven critical role for CD8+ T cells in protection against infectious diseases caused by intracellular microbes, effective vaccines that target this T cell subset currently do not exist. This is because naturally processed and presented CD8+ T cell epitopes and those that confer protective immunity remain unknown. To fill this gap, using the most successful smallpox vaccine as a model, we first discovered and characterized numerous HLA- A*02;01 and -B*07;01 class I restricted, naturally processed CD8+ T cell-epitopes derived from vaccinia virus (VacV) that are also conserved in variola virus-the agent of smallpox. We then found that prime-boost vaccination of mice with engineered VacV proteins containing such epitopes in combination with an NKT cell-targeted adjuvant - galactosylceramide (GC)-which rapidly activates NKT cells and potently trans-activates dendritic cells as well as natural killer, T and B cells-elicited robut, functional CD8+ T cell responses. These CD8+ T cell responses protected mice from lethal respiratory poxvirus challenge. Guided by these findings, we will test the central hypothesis that, Nanoparticle- based mucosal delivery of microbial protein subunit vaccines formulated with an optimal adjuvant confers protective immunity against lethal respiratory challenge by targeting naturally processed CD8+ T cell epitopes. Our approaches to test the central hypothesis are: (a) to prepare nanoparticles (np) coupled with GC (GC-np) or the IFNy-inducing GC variant, flurobenzyl-acyl-GC (FBzGC-np), and to characterize the mechanism of their action; (b) to determine the quality and robustness of pulmonary CD8+ T cell responses to immunization with nanoparticles that will co-deliver coupled recombinant VacV subunit(s)- np and GC-np or FBzGC-np; and (c) to elucidate the protective potential of such a vaccine strategy against lethal respiratory challenge with heterotypic (VacV-WR strain) and homotypic (Ectromelia) poxvirus models. The approaches proposed herein are feasible because our new data revealed that rVV-np+GC-np elicited better protective response in humanized mice upon intranasal vaccination than intra-peritoneal immunization. Hence, we have proposed innovative approaches to develop microbe-free, mucosal vaccines containing naturally processed antigens and NKT cell-targeted adjuvant, which when delivered on nanoparticle carriers, mediate protection through CD8+ T cells. Such mucosal vaccine formulation targets previously unemployed, yet greatly potent arms of the immune system critical for protection against intracellular infectious disease agents-viz., the innate-like NKT cells and the adaptive CD8+ T cells. Because the vaccine will be tested in both heterotypic and homotypic pathogen-host models and a humanized mouse model, we expect to gain novel insights relevant to the design of next generation pathogen-free vaccines. Such vaccines will have the potential to shift clinical practice paradigms, especially against infectious diseases that directly impact the health and well-being of this World's peoples, and, hence, will benefit Veterans as well.